US6017748A - Cloning host organisms - Google Patents
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- US6017748A US6017748A US08/488,423 US48842395A US6017748A US 6017748 A US6017748 A US 6017748A US 48842395 A US48842395 A US 48842395A US 6017748 A US6017748 A US 6017748A
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Definitions
- This invention relates to certain methods and materials for the cloning of DNA and, in particular, to the cloning of "unclonable" DNA using a genetically engineered host cell.
- This cloning host organism includes particular mutations from existing cells which have been found to stabilize tester DNA plasmids, and can be assembled using standard transducing and plating techniques.
- the ability to elucidate gene structure and function often depends to a great extent upon the construction of recombinant DNA libraries accurately representing the total genome of a subject organism, followed by the cloning of this recombinant DNA.
- the total DNA of cells from the subject organism is isolated and cut into fragments using specific restriction enzymes.
- the fragmented DNA sequences are then inserted into appropriate vectors for subsequent propagation and amplification in a foreign host.
- Commonly used vectors include plasmids, cosmids or phages.
- host cells suitable for propagation of foreign DNA sequences include bacteria (i.e., E.
- Drosophilia i.e., Drosophilia melongaster mammalian cell lines (CHO, L-cells) human cell lines (i.e., Hela, Baculovirus, plant cell lines.
- DNA that is capable of forming cruciforms (hydrogen bonded hairpin structures formed from inverted repeat sequences) and Z-DNA (a left handed zig zag configured DNA resulting from alternating purine-pyrimidine residues), for example, is rapidly deleted or rearranged in E. coli.
- cruciforms hydrogen bonded hairpin structures formed from inverted repeat sequences
- Z-DNA a left handed zig zag configured DNA resulting from alternating purine-pyrimidine residues
- the present inventor has discovered that complex DNA sequences can be stabilized during cloning by utilizing hosts with combined mutations in certain nucleic acid recombination and repair pathways. Described herein are novel bacterial hosts designed for cloning foreign DNA by combining DNA repair mutations into host strains deficient in homologous recombination. Mutations which allow for stabilization of tester plasmids containing complex DNA are identified in existing strains and then assembled using standard techniques of transduction, screening, selection and propagation. The resulting bacteria can be used to stabilize and clone complex DNA such as contained in eucaryotic genomes.
- This invention provides for the genetic construction of host organisms that provide a stable environment for cloning foreign DNA molecules capable of assuming secondary and tertiary structure susceptible of rearrangement. These hosts are, thus, suitable for cloning DNA molecules capable of assuming such secondary and tertiary structures.
- the host organisms of this invention provide a stable environment for DNA molecules containing inverted repeat sequences, capable of forming cruciforms, and DNA molecules containing alternating purine-pyrimidine residues, capable of forming Z-DNA.
- the host organisms of the invention are characterized as being recombination deficient and containing genetic mutations in DNA repair pathways.
- this invention provides for an E.
- the invention further provides for a method of cloning structurally complex DNA, such as found in the eucaryotic genome.
- This method comprises isolating DNA from an organism, cutting the DNA into fragments, inserting DNA fragments of that organism into a vector, and introducing the vector in the insert into the host organism of this invention.
- the method involves the use of a host characterized as recombination deficient with mutations in DNA repair systems.
- the E isolating DNA from an organism, cutting the DNA into fragments, inserting DNA fragments of that organism into a vector, and introducing the vector in the insert into the host organism of this invention.
- the method involves the use of a host characterized as recombination deficient with mutations in DNA repair systems.
- the E isolating DNA from an organism, cutting the DNA into fragments, inserting DNA fragments of that organism into a vector, and introducing the vector in the insert into the host organism of this invention.
- the method involves the use of a host characterized as recombin
- coli host used in cloning is of the genotype recB, recJ, sbcC201, phoR, uvrC, umuC::Tn5, mcrA, mcrB, mrr, ⁇ (hsdRMS), endA1, gyrA96, thi, relA1, lac*, supE44, ⁇ F'proAB, lacI Q Z ⁇ M15::Tn10 ⁇ .
- This invention further provides for a method of constructing a host organism suitable for cloning DNA molecules capable of assuming secondary and tertiary structures susceptible of rearrangement.
- the method comprises identifying mutations of recombination and DNA repair pathways which stabilize DNA in existing hosts and assembling these mutations using standard genetic techniques.
- This invention further provides for construction of a novel strain of E. coli, designated SURETM Construction of the SURETM strain is accomplished by successively transducing E. coli ER1451 with bacterial genomes containing mutations in recombination and DNA repair systems.
- a preferred method of constructing the SURETM strain is outlined in FIG. 2 and further described herein.
- FIG. 2 is a flow diagram of the steps used to construct a strain of E. coli that is suitable for cloning DNA capable of forming secondary and tertiary structures susceptible of rearrangement.
- the SURETM strain was assembled from ER1451 using sequential steps of P1 transduction (P1) Bochner plating, and conjugal mating.
- P1 transduction P1 Bochner plating, and conjugal mating.
- the E. coli strain designation for each P1 transduction step indicates the source of genetic material transduced.
- the genotypes of these strains are set forth in Table 1.
- FIG. 1 is a schematic representation of the recombination pathways of E. coli.
- FIG. 3 is a schematic gene map of the inverted repeat tester plasmid, designated pAL28 ⁇ .
- FIG. 4 is a schematic gene map of the Z-DNA tester plasmid system, designated pAL20Z.
- lac*--an uncharacterized mutation within the lac operon which results in colonies more intensely blue in the presence of the lacZ ⁇ M15 mutation and a source of the ⁇ -complementation lac segment
- infection--any process used to introduce DNA into host organisms Includes transduction, transformation transtection, conjugal mating, and electroporation.
- restriction minus--cell strains lacking genes coding for enzymes which break down foreign DNA
- DNA molecules capable of assuming nonstandard secondary and tertiary structures, and as commonly found in eucaryotic genomes, are often completely deleted or rearranged during propagation in cloning host cells.
- a major reason for this lack of fidelity in cloning may be the existence in the host cell of mechanisms related to the replication and repair of DNA. Examples of such mechanisms include: (1) pathways designed for homologous or non-homologous recombination; (2) restriction systems which cleave foreign DNA sequences; (3) endonucleases which catalyze the breakdown of DNA; and (4) systems designed for repair of DNA.
- procaryotic and eucaryotic organisms have developed an extensive array of systems designed for DNA repair.
- repair systems include: uv repair pathway; SOS repair pathway; mismatch repair; adaptive response; heat shock response; osmotic shock response; repair of alkylation damage; repair of uracil incorporation into DNA; and gene products involved in maintaining DNA superhelicity.
- the role of such repair systems in cloning of foreign DNA has not yet been investigated. As noted above, prior attempts to increase stabilization of foreign DNA have focused only on host cell mutations in the recombination and/or restriction pathways.
- the inventors have engineered a host organism with mutations in repair as well as recombination pathways.
- the resulting host cells serve to stabilize various complex forms of DNA during propagation.
- the new strain has been designed to be a superior host for optimizing the construction of cDNA and genomic DNA libraries as well as for increasing the probability that the individual clones obtained will accurately reflect the genetic makeup of the subject organism.
- the construction of host strains with combined mutations was accomplished by crossing or mating strains with individual mutations in metabolic pathways.
- Means of constructing new strains by DNA introduction or mutation include, but are not limited to, Hfr mating, generalized transduction, specialized transduction, conjugation, transposon mutagenesis, oligonucleotide-directed mutagenesis, and transformation followed by genetic recombination.
- transducing bacteriophages include, but are not limited to, P1 or lambda.
- Example 1 Details of a preferred method of construction using P1 phage transduction are presented in Example 1. Screening and identification of mutant strains incorporating and expressing the transduced foreign DNA can be accomplished by any number of methods. Examples of such identification and screening methods include, but are not limited to, screening for phenotypic traits such as antibiotic sensitivity, drug resistance, or dependency on nutrient sources.
- the parent strain used as the basis for construction, can be any existing strain with a well-defined genotype that preferably includes at least one of the specific mutations sought to be present in the final genotype. Numerous such strains are available and have been well characterized. See, e.g., Ishiura, M., Hazumi, N., Koide, T., Uchida, T., and Okada, Y. (1989) J. Bact. 171:1068-1074.
- the parent strain is then transduced with or mated with DNA from a second strain known to contain at least one additional desired mutation. Those progeny cells containing the desired mutations from both the donor and recipient cell are identified, propagated and used through subsequent transduction or mating steps.
- the invention is a propagatable cell containing combined mutations in both recombination and DNA repair systems.
- the cloning host cell also contains mutations in the endonuclease and restriction systems, which mutations increase the utility of host cells for cloning purposes.
- the inventors have engineered and constructed a novel strain of E. coli, with combined mutations in recombination and repair systems, that stabilizes complex foreign DNA during propagation.
- E. coli strain ER1451 known to have mutations of certain DNA endonucleases and components of the restriction pathway, was sequentially transduced with bacterial genomes having the mutations sought to be introduced. In each case, the transduced DNA also contained a gene for tetracycline resistance. Following each individual transduction step, strains containing the combined mutations were screened on Bochner plates in order to isolate tetracycline sensitive derivatives which would then serve as a recipient for the next transduction step. Successive transduction steps can be performed in any order.
- the ER1451 strain was transduced sequentially with DNA containing mutations in the recombination pathway, ultraviolet repair pathway, SOS repair pathway, the restriction pathway, a tetracycline-resistant F'episome carrying a blue/white screening cassette and a lactose sensitivity gene.
- a strain of bacteria was created that is recombination deficient, restriction minus, endonuclease deficient, and with mutations in DNA repair systems. Details of this preferred construction sequence are given in Example 1.
- the resulting bacterial host, constructed by this method, is known as SURE, a trademark of Stratagene Cloning Systems.
- the SURETM strain constructed by this method was then tested to determine its effect on the stability of cloned DNA sequences capable of forming secondary and tertiary structures and susceptible to rearrangement during propagation.
- DNA sequences include, but are not limited to inverted repeats and alternating purine-pyrimidine residues.
- inverted repeat plasmid tester system was created by modifying plasmid pBR322. This tester plasmid system, designated pAL28 ⁇ , used chloramphenicol resistance as the inverted repeat sequence.
- pAL28 ⁇ This tester plasmid system, designated pAL28 ⁇ , used chloramphenicol resistance as the inverted repeat sequence.
- a variety of strains of E. coli, existing strains as well as the SURETM strain were infected with a tester inverted repeat plasmid. Inverted repeats were found to be rapidly rearranged in virtually all existing strains tested. In contrast to the results with existing strains, however, the SURETM strain was found to provide a stabilizing environment for these inverted repeat sequences.
- the percentage of rearrangement was decreased 20 fold in the SURETM strain when compared to the other strains tested.
- the SURETM strain was, therefore, engineered to carry mutations blocking pathways responsible for repairing DNA lesions.
- Preferred specific mutations for blocking these two key pathways are uvrC and umuC respectively, although mutations in other genes of this pathway or mutations in other repair pathways would give the desired effect. This presence of these mutations was demonstrated to result in a 10 to 20 fold increase in stability of DNA containing long inverted repeats.
- Z-DNA tester plasmids were created as derivatives of plasmid pBR322.
- the pBR322 plasmid containing sequences for tetracycline and penicillin resistance, was modified to include a Z-DNA segment in the promoter region for chloramphenicol resistance.
- the newly created tester plasmid system was designated pAL20Z.
- This tester DNA plasmid was infected into existing strains of E. coli as well as the newly constructed SURETM strain.
- the Z-DNA containing segments were quickly deleted in the existing strains while, in the SURETM strain, deletion was significantly and markedly reduced.
- homologous recombination is a complex, multicomponent process that can proceed by three interdependent pathways (see FIG. 1). These 3 pathways, recBCD, recE and recF, all require the recA gene product and a set of accessory proteins.
- a standard recA host therefore, is completely deficient in homologous recombination.
- Recombination in a recb strain (the recBCD pathway is the primary pathway in a wild type E. coli) is reduced to approximately 0.5%.
- the residual activity is due to the presence of the recE and recF alternate pathways which do not involve recB.
- a recB recJ double mutant is virtually identical to a recA strain in its recombination deficiency; recJ is required for both alternate pathways.
- recombination can be blocked in host organisms with a variety of mutations. In addition to the ones set forth above, examples of such alternate mutations include recB, reco and recB, recJ, recN.
- the inventors discovered that insertion of either the recA or recB mutations into a sbcC, recJ, umuC, uvrC strain, stabilized both Z-DNA and inverted repeats.
- the recB derivative was found to exhibit superior stabilization effects. This discovery, that combined mutations in recombination and DNA repair pathways, stabilize complex DNA during propagation is significant.
- the cloning host organism of the instant invention is restriction minus, preferably carrying, in E. coli, hsd, mcr and mrr mutations.
- the absence of mcr and mrr restriction activity increases the size and representation of libraries constructed with methylated or hemimethylated DNA.
- the absence of hsd activity increases the size and representation of libraries constructed from sequences containing EcoK recognition sequences.
- An end mutation inactivating a DNA endonuclease was observed to result in mproved quality of plasmid DNA minipreps.
- the host cell of this invention also harbors a tetracycline-resistant F'episome carrying the lacI Q Z ⁇ M15 cassette making it suitable for blue/white screening on plates supplemented with X-gal and IPTG.
- the uncharacterized mutation present in XL1-Blue cells which makes both plaques and colonies more intensely blue when a source of the ⁇ -complementation fragment is introduced, has been added.
- the cell is, thus, suitable for plasmid or phage libraries using a variety of vectors.
- a novel strain of E. coli was constructed from existing strains utilizing standard transduction and plating techniques. In this example, transduction with P1 phage particles was utilized. Bochner plating was then used to screen derivatives for the desired genotype.
- An outline of the construction method is schematically diagramed in FIG. 2. The genotypes of the various E. coli strains used in the construction pathway are set forth in Table 1.
- ER1451 cells were grown at 37° C. to a density of 5 ⁇ 10 8 to 1 ⁇ 10 9 cells/ml in media containing tryptone, yeast extract, NaCl and agar (LB media) containing 5 mM CaCl 2 .
- Cells were harvested by centrifugation and resuspended in 100 mM MgSo 4 , 5 mM CaCl 2 (MC media) media at 37° C. for 15 minutes. Cells were again harvested by centrifugation and resuspended in 1/10 the original volume of MC media.
- 0.1 ml of the resulting cell suspension was mixed with a P1 phage, containing the genome of E. coli strain CES229, and incubated at 37° C. for 15 minutes.
- the reaction was terminated by the addition of 0.1 ml of 0.2M sodium citrate. 0.5 ml of LB media was added and cultures grown at 37° C. for an additional 60 minutes. These incubation conditions were found to be sufficient for expression of transduced DNA. Cells (5 ⁇ 10 6 ) were then plated directly onto LB plates containing 15 ⁇ g/ml tetracycline. Transductants were screened for the desired phenotype, then plated onto Bochner plates and screened for tetracycline sensitivity.
- the culture media for this screening procedure contained in final concentration: 10 g/L tryptone, 5 g/L yeast extract, 15 g/L agar, 10 g/L NaCl, 10 g/L NaH 2 PO 4 , 2 g/L glucose, 12 mg/L fusaric acid, 0.1 mM ZnCl 2 , and 50 mg/L of chlortetracycline.
- Cell colonies demonstrating a rapid rate of growth and sensitivity to tetracycline were identified, recovered and utilized for subsequent transduction as indicated in FIG. 2.
- ER1451 cells were sequentially transduced with P1 phages containing the genome of E.
- SURETM a trademark of Stratagene Cloning Systems.
- the genotype of SURETM is recB, recJ, sbcC201, phoR, uvrC, umuC::Tn5, mcrA, mcrB, mrr, ⁇ (hsdRMS), endA1, gyrA96, thi, relA1, lac*, supE44, ⁇ F'proAB, lacI Q Z ⁇ M15, Tn10 ⁇ .
- SURETM strain of E. coli was then used to test for stability of various structurally complex DNA sequences.
- DNA molecules containing an inverted repeat sequence coding for chloramphenicol resistance were created and inserted into modified pBR322 plasmid vectors.
- a gene map of the inverted repeat tester system, designated plasmid pAL28 ⁇ , is schematically diagramed in FIG. 3. Numerous strains of E. coli, including the SURETM strain, were transformed with pAL28 ⁇ . Transformants were inoculated in media containing only penicillin and grown for up to 100 generations (with serial dilutions). After every 10 or 20 generations, aliquots of cells were screened on either penicillin containing or chloramphenicol containing plates. Cells in which the inverted repeat chloramphenicol resistant sequences were rearranged lost their chloramphenicol resistance.
- DNA molecules were created which contained alternating purine and pyrimidine residues capable of forming Z-DNA. These Z-DNA containing sequences were inserted into a modified pBR322 plasmid designated pAL20Z (see FIG. 4). A sequence of 26 alternating purine-pyrimidine residues (GC) were inserted into and blocked expression of the promoter region for chloramphenicol resistance. Because of the absence of a promoter in the intact plasmid, host cells containing pAL20Z demonstrate chloramphenicol sensitivity. Deletion of the repeating sequences results in reconstitution of the promoter and, therefore, expression of chloramphenicol resistance. A variety of E. coli host strains were transformed with pAL20Z.
- GC purine-pyrimidine residues
- Transformants were inoculated into media containing penicillin and grown for up to 200 generations (with serial dilutions). After every 20 generations, 10 6 cells were plated onto media containing chloramphenicol (50 ⁇ g/ml). The percentage of cells demonstrating chloramphenicol resistance was taken as a direct reflection of the percent of cells rearranging the tester plasmid.
- the results from transformation of seven of these E. coli strains, including the SURETM strain, are shown below in Table 3. The genotypes of these E. coli strains can be found in Table 1.
- the SURETM E. coli strain significantly and markedly reduced the amount of DNA rearrangement.
- the SURETM strain therefore, provides a stabilizing environment for Z-DNA.
- the data from Examples 2 and 3 demonstrate that a cloning host organism can be engineered and constructed such that it provides a stabilizing environment for complex DNA molecules capable of forming secondary and tertiary structures.
- this host organism is effective in inhibiting rearrangements of DNA molecules capable of forming a variety of such secondary and tertiary structures.
- a novel strain of bacteria has been engineered by combining a number of mutations which significantly reduce the rate of both homologous and non-homologous recombination within segments of cloned foreign DNA.
- Non-bacterial DNA particularly from eucaryotic sources, frequently contain sequences capable of forming secondary and tertiary structures such as cruciforms (due to presence of inverted repeats) or Z-DNA (found in alternating purine-pyrimidine stretches). These structures are highly recombinogenic in standard bacterial cloning hosts which are incapable of carrying out homologous recombination. Mutations which eliminate certain repair pathways responsible for excision of or replication past DNA lesions greatly stabilize these unusual structures. By engineering mutations which eliminate these pathways into a host strain deficient in homologous recombination, a novel host that is suitable for cloning foreign DNA has been constructed.
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Abstract
Description
TABLE # 1 ______________________________________ Genotypes Strain ______________________________________ ER1451 Δ(lac-proAB), thi, gyrA96, endA1, hsdR17, relA1, supE44, {F' traD36, proAB, lacI.sup.Q Z M15} CES229 recD1009, hsdR, sbcC201, phoR79::Tn10, recA JC12166 recB21, recC22, sbcB15, sbcC201, thr-1, leuB6, thi-1, lacY1, galk2, ara-14, xyl-5, mtl-1, proA2, his-4, argE3, rpsL31, tsx-33, sup44, recJ284::Tn10 CAG12156 MG1655 (E. coli K-12 F.sup.- ) uvrC279::Tn10 JC8947 AB1157 (standard E. coli K-12) umcC::Tn5 AG279 same as JC12166 except recJ.sup.+, recF143, fuc::Tn10 JH122 GW1000 (standard E. coli K12, mcrB4::Tn10, mrr2::Tn5, hsdR2) BB4 hsdR514, supE44, supP58, lacY1, galK2, galT22, metB1, trpR55, {F'(lacI.sup.Q Z M15::Tn10} AG239 thi, gyrA, supE44, lac.sup.- (uncharacterized) Tet.sup.R AG1 recA, endA1, gyrA96, thi, hsdR17, supE44, relA C600· supE44, thi-1, leuB6, lacY1, tonA21, mcrA NM621· recD, hsdR, mcrA, mcrB, supE HB101 hsdS20, supE44, ara14, galK2, lacY1, proA2, rpsL20, xyl-5, recA13, mcrB TBI· ara, (lac-proAB), rpsL, 80, lacZ M15, hsdR DH5α recA1, endA1, relA1, thi, hsdR17, supE44, gyrA96 ______________________________________
TABLE # 2 ______________________________________ STABILITY OF INVERTED REPEATS IN VARIOUS E. COLI STRAINS Percentage of cells generating rearranged Strain plasmid per generation.sup.a ______________________________________ AG1 15-20 DH5α 15-20 C600 20-25 NM621 20-25 HB101 15-20 TB1 20-25 DL538 20-25 SURE ™ 0.8-1.2 ______________________________________ .sup.a Percent rearrangement calculated from the number of cells maintaining the inverted repeat tester plasmid in its unrearranged configuration (chloramphenicol resistant) divided by the total number of plasmidcontaining cells.
TABLE # 3 ______________________________________ STABILITY OF Z-DNA IN VARIOUS E. COLI STRAINS Percentage of cells generating rearranged Strain plasmids per 25 generations.sup.a ______________________________________AG1 15DH5α 15 C600· 10 NM621· 20HB101 15 TB1· 10 SURE ™ strain <0.1 ______________________________________ .sup.a Percent ZDNA rearrangement calculated by determining the percentag of chloramphenicol resistant cells (indicative of a specific ZDNA rearrangement) within a population after 25 generations.
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Cited By (4)
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US20030123883A1 (en) * | 1998-11-04 | 2003-07-03 | The Research Foundation Of State University Of New York | Method and apparatus for detecting radiation |
WO2005121351A2 (en) | 2004-05-27 | 2005-12-22 | Novozymes, Inc. | Methods for transforming and expression screening of filamentous fungal cells with a dna library |
US20090203125A1 (en) * | 2006-05-02 | 2009-08-13 | Washington State University | Mig-7 as a specific anticancer target |
WO2018094181A1 (en) | 2016-11-21 | 2018-05-24 | Novozymes A/S | Yeast cell extract assisted construction of dna molecules |
Families Citing this family (11)
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ATE201047T1 (en) * | 1990-01-08 | 2001-05-15 | Stratagene Inc | NEW HOST ORGANISMS FOR CLONING |
US6720140B1 (en) * | 1995-06-07 | 2004-04-13 | Invitrogen Corporation | Recombinational cloning using engineered recombination sites |
US6143557A (en) * | 1995-06-07 | 2000-11-07 | Life Technologies, Inc. | Recombination cloning using engineered recombination sites |
CN101125873A (en) * | 1997-10-24 | 2008-02-20 | 茵维特罗根公司 | Recombinational cloning using nucleic acids having recombination sites |
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US20030123883A1 (en) * | 1998-11-04 | 2003-07-03 | The Research Foundation Of State University Of New York | Method and apparatus for detecting radiation |
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US20090203125A1 (en) * | 2006-05-02 | 2009-08-13 | Washington State University | Mig-7 as a specific anticancer target |
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EP0437071A1 (en) | 1991-07-17 |
DE69033732T2 (en) | 2001-10-11 |
JPH04211361A (en) | 1992-08-03 |
CA2033355A1 (en) | 1991-07-09 |
EP0681025A1 (en) | 1995-11-08 |
EP0681025B1 (en) | 2001-05-09 |
US5552314A (en) | 1996-09-03 |
DE69033732D1 (en) | 2001-06-13 |
ATE201047T1 (en) | 2001-05-15 |
CA2033355C (en) | 2001-07-24 |
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